Appl Microbiol Biotechnol (2006) 71: 394–406 DOI 10.1007/s00253-006-0427-1
MINI-REVIEW
Kirsi Savijoki . Hanne Ingmer . Pekka Varmanen
Proteolytic systems of lactic acid bacteria
Received: 30 November 2005 / Revised: 13 March 2006 / Accepted: 13 March 2006 / Published online: 21 April 2006 # Springer-Verlag 2006
Abstract Lactic acid bacteria (LAB) have a very long history of use in the manufacturing processes of fermented foods and a great deal of effort was made to investigate and manipulate the role of LAB in these processes. Today, the diverse group of LAB includes species that are among the best-studied microorganisms and proteolysis is one of the particular physiological traits of LAB of which detailed knowledge was obtained. The proteolytic system involved in casein utilization provides cells with essential amino acids during growth in milk and is also of industrial importance due to its contribution to the development of the organoleptic properties of fermented milk products. For the most extensively studied LAB, Lactococcus lactis, a model for casein proteolysis, transport, peptidolysis, and regulation thereof is now established. In addition to nutrient processing, cellular proteolysis plays a critical role in polypeptide quality control and in many regulatory circuits by keeping basal levels of regulatory proteins low and removing them when they are no longer needed. As part of the industrial processes, LAB are challenged by various stress conditions that are likely to affect metabolic activities, including proteolysis. While environmental stress responses of LAB have received increasing interest in recent years, our current knowledge on stressrelated proteolysis in LAB is almost exclusively based K. Savijoki . P. Varmanen Department of Basic Veterinary Sciences, Faculty of Veterinary Medicine, University of Helsinki, P.O. Box 66, Helsinki 00014, Finland K. Savijoki (*) Institute of Biotechnology, University of Helsinki, P.O. Box 56, Helsinki 00014, Finland e-mail:
[email protected] Tel.: +358-9-19159414 H. Ingmer Department of Veterinary Pathobiology, The Royal Veterinary and Agricultural University, Stigbøjlen 4, Frederiksberg C 1870, Denmark
on studies on L. lactis. This review provides the current status in the research of proteolytic systems of LAB with industrial relevance.
Introduction Lactic acid bacteria (LAB), including members of the genera Lactobacillus, Lactococcus, Leuconostoc, Pediococcus, and Streptococcus, are defined as gram-positive, nonsporulating, catalase-negative, and facultative anaerobic bacteria with a fermentative metabolism (Axelsson 1998). LAB were found to have applications in manufacturing various fermented foods, beverages, and feed products (Leroy and Devuyst 2004). In addition, certain LAB strains, most notably the strains from the genera Lactobacillus, are increasingly marketed as health-promoting, i.e., probiotic bacteria (Saxelin et al. 2005), while certain Lactobacillus strains are believed to produce bioactive health-beneficial peptides from milk proteins (Meisel and Bockelman 1999; Korhonen and Pihlanto 2003). Another promising avenue for LAB is to use them as delivery vehicles for molecules with therapeutic value (Nouaille et al. 2003). Lactococcus lactis is the most extensively studied LAB organism and the second most studied gram-positive bacterium with respect to its genetics, physiology, and molecular biology. Undoubtedly, the most important application of LAB is their use as starter strains in the manufacture of various fermented dairy products. In particular, Streptococcus thermophilus, L. lactis, Lactobacillus helveticus, and Lactobacillus delbrueckii subsp. bulgaricus (hereafter L. bulgaricus) are widely used dairy starters and are of major economic importance. In the milk fermentation processes, the proteolytic system of LAB plays the key role because it enables these bacteria to grow in milk, thereby ensuring successful fermentation. LAB are fastidious microorganisms that require an exogenous source of amino acids or peptides, which are provided by the proteolysis of casein, the most abundant protein in milk and the main source of amino acids. In general, the exploitation of casein by LAB is initiated by a cell-envelope proteinase (CEP) that
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degrades the protein into oligopeptides that are subsequently taken up by the cells via specific peptide transport systems for further degradation into shorter peptides and amino acids by a concerted action of various intracellular peptidases (Kunji et al. 1996; Christensen et al. 1999). While many LAB strains contain CEP, several of these strains, i.e., nonstarter LAB, do not, thus, they rely on starter LAB for the production of peptides and amino acids. These pathways are also of industrial importance because in addition to allowing growth, peptides, amino acids, and their derivatives are also known to contribute to the formation of texture and flavor of the fermented milk products. Therefore, a number of studies were undertaken to unravel the pathways underlying this industrially relevant trait and excellent reviews have covered these aspects of the most extensively utilized dairy starters (Kok and de Vos 1994; Poolman et al. 1995; Kunji et al. 1996; Mierau et al. 1997; Christensen et al. 1999; Doeven et al. 2005). The proteolytic system of L. lactis was investigated to the extent that a complete model for casein breakdown, transport, degradation of casein-derived peptides, and regulation thereof was established (Tynkkynen et al. 1993; Hagting et al. 1994; Foucaud et al. 1995; Detmers et al. 1998; Guédon et al. 2001a,b; den Hengst et al. 2005a, b). In addition to this “casein exploitation machinery,” LAB, like any other cell, encode stress-inducible proteolytic enzymes that are active on casein in vitro, but are in vivo known to be responsible for the processing of abnormal proteins created by, e.g., heat shock (Frees and Ingmer 1999). To date, the published genome sequences of starter LAB include L. lactis (Bolotin et al. 2001), S. thermophilus (Bolotin et al. 2004), and Lactobacillus sakei (Chaillou et al. 2005). Three other genome sequences were published for probiotic LAB, Lactobacillus plantarum (Kleerebezem et al. 2003), Lactobacillus acidophilus (Altermann et al. 2005), and Lactobacillus johnsonii (Pridmore et al. 2004). Furthermore, several other whole genome sequences of LAB were recently completed and are expected to be published in the near future (Klaenhammer et al. 2005). Comparative genomics reveals some differences between the proteolytic systems of LAB; differences that are thought to reflect the various environmental niches these bacteria occupy (Boekhorst et al. 2004). For example, of the sequenced lactobacilli, only L. sakei and L. johnsonii are equipped with CEPs. Compared to L. lactis, lactobacilli are largely deficient in amino acid biosynthetic capacity, which is compensated by an ability to encode a large number of peptidases, amino acid permeases, and multiple oligopeptide transport (Opp) systems (Klaenhammer et al. 2005). This review provides an overview of the components comprising the proteolytic system in LAB that are associated with food fermentations, placing special emphasis on components of the proteolytic system that were genetically and biochemically characterized. Recent advances in our understanding of the mechanisms employed by LAB to regulate proteolysis and the results of genetic engineering studies aiming to improve strain characteristics
in terms of proteolytic activity are also included in this review. High throughput “omics” technologies have also emerged in the field of dairy research and studies providing novel information relevant to casein utilization are discussed.
Proteinases The first step in casein utilization by LAB is performed by CEPs. Five different types of these enzymes were cloned and characterized from LAB, including PrtP from L. lactis and Lactobacillus paracasei, PrtH from L. helveticus, PrtR from Lactobacillus rhamnosus, PrtS from S. thermophilus, and PrtB from L. bulgaricus (Kok et al. 1988; Holck and Naes 1992; Gilbert et al. 1996; Pederson et al. 1999; Siezen 1999; Fernandez-Espla et al. 2000; Pastar et al. 2003). In lactococci, prtP genes can either be plasmid- or genomeencoded, whereas CEPs characterized from lactobacilli are genome-encoded. LAB typically possess only one CEP but the presence of two CEPs was reported in strains of L. helveticus and L. bulgaricus (Stefanitsi et al. 1995; Pederson et al. 1999). The CEPs of dairy LAB are typically synthesized as preproproteins of approximately 2,000 residues and are composed of several distinct functional domains (Fernandez-Espla et al. 2000; Siezen 1999) (Fig. 1). From the N terminus the CEPs include: the preprodomain (PP) corresponding to a signal sequence (∼40 residues) required for secretion and a prosequence (∼150 residues) that is removed by autocatalytic processing; the catalytic serine protease domain (PR) (∼500 residues); an insert domain (I) (∼150 residues) that possibly modulates the substrate specificity of CEPs; the A domain (∼400 residues) of unknown function; the B domain (∼500 residues) possibly involved in stabilizing the CEP activity/specificity; the helix domain (H) (∼200 residues) involved in positioning the A and B domains outside the bacterial cell; and a hydrophilic W domain (∼100 residues) functioning as a cell wall spacer. While the B domain is found in most CEPs, it is not present in the PrtS of S. thermophilus. The W domain of PrtS contains the typical amino acid composition of the cell wall domain of gram-positive bacteria (rich with Pro-Gly and SerThr); however, it has no homologies with CEPs or with any other proteins from the database (Fernandez-Espla et al. 2000). In contrast, the H domain is present only in PrtP (210 aa), PrtS (367 aa), and PrtH (72 aa). In PrtP and PrtS, the W domain is followed by a cell wall anchor domain (AN) carrying a sorting signal typical for many surface proteins of gram-positive bacteria (Navarre and Schneewind 1994). The L. helveticus and L. bulgaricus CEPs lack the AN domain and are instead thought to bind the cell wall using the W domain itself (Pederson et al. 1999; Siezen 1999). In L. helveticus, PrtH is possibly attached to the cell wall via a mechanism resembling that utilized by S-layer proteins (Pederson et al. 1999; Leenhouts et al. 1999). The PrtR
396 Fig. 1 Schematic representation of CEPs of different LAB strains (model according to Siezen 1999). CW Cell wall, M membrane, C cytoplasm, PP prepro domain, PR catalytic domain, I insert domain, A A domain, B B domain, H helix domain, W cell wall spacer domain, black dot sorting signal, and AN anchor domain
CW M C
I
L. lactis L. paracasei PrtP
PP
PR
A
B
H
W
AN
I L. helveticus PrtH
PP
PR
A
B
H W
I L. bulgaricus PrtB
PP
PR
A
B
PR
A
H
W
I S. thermophilus PrtS L. rhamnosus PrtR
characterized from L. rhamnosus BGT10 is markedly different from the CEPs of starter LAB (Pastar et al. 2003). The B domain of PrtR is somewhat smaller and different in that it lacks the helix and insert domains; its W domain is more homologous to certain cell-surface antigens expressed by oral and vaginal streptococci and its AN domain contains an atypical sorting signal. In L. lactis, the prtP gene is preceded by a divergently transcribed gene encoding a membrane-bound lipoprotein (PrtM) that was shown to be essential for autocatalytic maturation of PrtP (Haandrikman et al. 1989, 1991). The same genetic organization for the two genes was demonstrated for L. paracasei (Holck and Naes 1992), whereas in L. helveticus, L. bulgaricus, or S. thermophilus, no prtM gene was identified in the regions flanking the genes encoding CEPs (Fernandez-Espla et al. 2000; Siezen 1999). It was reported that the maturation process of CEPs of L. bulgaricus does not require a PrtM-like chaperone (Gilbert et al. 1996; Germond et al. 2003). CEPs have a strong preference for hydrophobic caseins, the most abundant proteins in milk (Swaisgood 1982). Caseins are divided into the αs1-, αs2-, β-, and κ-caseins; each contains a large number of proline residues that both prevents the formation of α-helices and β-sheets and promotes the formation of random coils. Together, these secondary structure characteristics lead to an unstructured, open molecule susceptible to action of CEPs (Fox 1989a). Lactococcus PrtPs are divided into PI- and PIII-type enzymes, distinguished by their substrate specificity for αS1-, β-, and κ-caseins (Kunji et al. 1996). The PI-type primarily degrades β-casein that is cleaved into more than 100 different oligopeptides ranging from 4 to 30 amino acid residues (Juillard et al. 1995). κ-casein is cleaved to a lesser extent by the PI-type enzyme, whereas the PIII-type is able to cleave αS1-, β−, and κ-caseins equally well (Pritchard and Coolbear 1993). PrtPs are further classified into seven groups (a, b, c, d, e, f, and g) by their specificity toward the αS1-casein fragment from positions 1 to 23 (f1–23) (Kunji et al. 1996). PrtP of L. lactis was also shown to act on autolysin (Buist et al. 1998), which is required for cell separation and autolysis during the stationary phase of growth. The rate and degree of autolysis was found to depend on the specificity, location, and the amount of the
PP
PP
PR
A
B
W
AN
W
AN
PrtP. For Lactobacillus, CEPs PI-, PIII-, the intermediate PI/PIII-type, and some novel type substrate specificities were reported, whereas a CEP (PrtS) exhibiting the intermediate PI/PIII-type specificity was purified from S. thermophilus (Fernandez-Espla et al. 2000).
Peptide uptake systems The second step in casein utilization includes transportation of peptides generated by CEP into the cell by the action of the Opp system; this was recently covered in depth in another review (Doeven et al. 2005) and is therefore only briefly discussed here. The Opp proteins belong to a superfamily of highly conserved ATP-binding cassette transporters that mediate the uptake of caseinderived peptides (Higgins 1992). In L. lactis MG1363, genes encoding the oligopeptide-binding protein (OppA), two integral membranes (OppB and OppC) and the nucleotide-binding (OppD and OppF) proteins exist in an operon (Tynkkynen et al. 1993). The Opp system of L. lactis transports peptides up to at least 18 residues and the nature of these peptides significantly affects the transport kinetics involved (Detmers et al. 1998; Juillard et al. 1998). Generally, the Opp systems of other LAB are not widely investigated but it was reported that in S. thermophilus (Garault et al. 2002) and L. bulgaricus (Peltoniemi et al. 2002), the composition of the Opp system is similar to that described for Lactococcus. It should be noted that S. thermophilus carries three paralogs of genes encoding OppA proteins, each of which is involved in oligopeptide internalization (Garault et al. 2002). Other peptide transporters identified in L. lactis MG1363 and IL1403 strains include a proton motive force (PMF)-driven dipeptide/tripeptide DtpT and an ATPdriven Dpp (previously referred to as DtpP) system (Hagting et al. 1994; Foucaud et al. 1995). Dpp is capable of transporting di-, tri-, and tetrapeptides containing relatively hydrophobic branched-chain amino acids (BCAAs) and displays the highest affinity for tripeptides (Foucaud et al. 1995; Sanz et al. 2003), whereas DtpT has a preference for more hydrophilic and charged di- and tripeptides (Hagting et al. 1994). L. helveticus also
397 Table 1 Peptidases genetically characterized from LAB Peptidase Endopeptidases PepO
LAB strain
Typea Substrate specificityb
L. lactis P8-2-47 L. lactis SSL135 L. helveticus CNRZ32 L. rhamnosus HN001 L. lactis IL1403/NCDO763 L. lactis NCDO763 L. lactis IL1403/NCDO763 L. helveticus CNRZ32 L. helveticus CNRZ32 L. helveticus CNRZ32 L. helvetiucs CNRZ32 L. helvetiucs CNRZ32 L. delbrueckii subsp. lactis DSM7290 S. thermophilus A
M M M M M M M M M C C M C M
NH2–Xn↓Xn–COOH
PepO2 PepF1 PepF2 PepO2 PepO3 PepE PepE2 PepF PepG PepO Aminopeptidases PepN L. lactis Wg2 M L. lactis MG1363 M L. delbrueckii subsp. lactis DSM7290 M L. helveticus CNRZ32 M L. helveticus 53/7 M S. thermophilus A M PepC L. lactis AM2 C L. delbrueckii subsp. lactis DSM7290 C L. helveticus CNRZ32 C L. helveticus 53/7 C S. thermophilus A C Aminopeptidases PepS S. thermophilus A M PepA L. lactis FI1876 M PepL Tripeptidases PepT Dipeptidases PepD PepV Proline-spesific PepQ
PepI
PepR
Reference
Mierau et al. 1993 Tynkkynen et al. 1993 Chen and Steele 1998 Christensson et al. 2002 Nardi et al. 1997 Monnet et al. 1994 Nardi et al. 1997 Chen et al. 2003 Sridhar et al. 2005 Fenster et al. 1997 Sridhar et al. 2005 Sridhar et al. 2005 Klein et al. 1997 Chavagnat et al. 2000 NH2–X↓Xn–COOH
NH2–X↓Xn–COOH
NH2–Glu/Asp↓Xn– COOH
L. delbrueckii subsp. lactis DSM7290 S
Strøman 1992 Tan et al. 1992 Klein et al. 1993 Christensen et al. 1995; Dudley and Steele 1994 Varmanen et al. 1994 Chavagnat et al. 1999 Chapot-Chartier et al. 1993 Klein et al. 1994a Fernández et al. 1994 Vesanto et al. 1994 Chapot-Chartier et al. 1994 Fernandez-Espla and Rul 1999 I’Anson et al. 1995 Klein et al. 1995
NH2–X↓X–X–COOH L. lactis MG1363 L. helveticus 53/7
M M
L. helveticus 53/7 L. helveticus CNRZ32 L. lactis MG1363 L. delbrueckii subsp. lactis DSM 7290
C C M M
L. L. L. L. L. L. L. L. L.
M M M S S S S S S
Mierau et al. 1994 Savijoki and Palva 2000 NH2–X↓X–COOH
delbruecki subsp. lactis DSM7290 bulgaricus B14 bulgaricus CNRZ 397 bulgaricus CNRZ397 delbrueckii subsp. lactis DSM7290 helveticus 53/7 helveticus CNRZ32 helveticus 53/7 rhamnosus 1/6
Vesanto et al. 1996 Dudley et al. 1996 Hellendoorn et al. 1997 Vongerichten et al. 1994 NH2–X↓Pro–COOH
NH2–Pro↓Xn–COOH
NH2–Pro↓X–COOH
Stucky et al. 1995 Rantanen and Palva 1997 Morel et al. 1999 Gilbert et al. 1994; Atlan et al. 1994 Klein et al. 1994b Varmanen et al. 1996a Dudley and Steele 1994; Shao et al. 1997 Varmanen et al. 1996b Varmanen et al. 1998
398 Table 1 (continued) Peptidase PepX
PepP
LAB strain
Typea Substrate specificityb
L. lactis NCDO763 L. delbrueckii DSM7290 L. helveticus CNRZ32 L. helveticus 53/7 L. rhamnosus 1/6 S. thermophilus ACA-DC4 L. lactis
S S S S S S M
Reference
NH2–X–Pro↓Xn–COOH Nardi et al. 1991 Meyer-Barton et al. 1993 Yüksel and Steele 1996 Vesanto et al. 1995 Varmanen et al. 2000a Anastasiou et al. 2002 NH2–X↓Pro–Xn–COOH Matos et al. 1998
a
Catalytic class of peptidase according to sequence analysis or biochemical characterization The arrow indicates the cleavage site M Metallopeptidase, C cysteine-peptidase, and S serine peptidase b
possesses a gene encoding a PMF-driven DtpT whose transport specificity resembles its lactococcal counterpart (Nakajima et al. 1997).
Peptidases A comprehensive review of the known specificities of LAB peptidases was published by Christensen et al. (1999). A
Casein-derived peptides (4-18 amino acid residues)
B
PepO
Free amino acids
PepI
Ile
PepQ
PepL
PepP
PepX
PepT PepR
PepV
Leu
C
CW M C
PepF
PepN PepC
DtpT
Peptides (2-4 amino acid residues)
Opp
PrtP
PrtM
Casein molecules
Dpp
A
large number of these enzymes were purified and biochemically characterized from S. thermophilus and different Lactococcus and Lactobacillus strains; in most cases, the corresponding gene was cloned and sequenced (Table 1). To date, no enzyme with carboxypeptidase activity was reported for any LAB. After the casein-derived peptides are taken up by the LAB cells, they are degraded by a concerted action of peptidases with differing and partly overlapping specific-
PepD
Val
CodY
codY
prtM
prtP
Fig. 2 Simplified presentation of the function and regulation of the proteolytic system of lactococci in casein breakdown (data from Kunji et al. 1996; Christensen et al. 1999; Doeven et al. 2005). a PrtP Cell-envelope proteinase, Opp oligopeptide permease, DtpT the ion-linked transporter for di- and tripeptides, and Dpp the ABC transporter for peptides containing 2 to 9 amino acid residues. b Intracellural peptidases. PepO and PepF are endopeptidases,
opp-pepO1
dtpT
pepN/C/X/DA2
PepN/PepC/PepP general aminopeptidases, PepX X-prolyl dipeptidyl aminopeptidase, PepT tripeptidase, PepQ prolidase, PepR prolinase, PepI proline iminopeptidase, and PepD and PepV dipeptidases D and V. c The transcriptional repressor CodY senses the internal pool of branched-chain amino acids (isoleucin, leucine, and valine); using these residues as cofactors CodY represses the expression of genes comprising the proteolytic system in L. lactis
399
ities (Kunji et al. 1996) (Fig. 2). The intracellular endopeptidases, general aminopeptidases (PepN and PepC), and the X-prolyl dipeptidyl aminopeptidase (PepX) are the first enzymes to act on oligopeptides. Several endopeptidases were characterized from LAB and assigned a variety of names (Table 1). All are metallopeptidases with the exception of the L. helveticus PepE, which was shown to exhibit a thiol-dependent activity (Fenster et al. 1997). A common feature of endopeptidases is their inability to hydrolyze intact casein but has the ability to hydrolyze internal peptide bonds of caseinderived peptides. For example, the αs1-casein f1–23 and/or β-casein f193–209 are the most preferred substrates for the endopeptidases of starter LAB origin. A unique cleavage specificity on αs1-casein f1–23 and on postproline residues of β-casein f203–209 was recently demonstrated for a PepO from a nonstarter strain, L. rhamnosus HN001 and for PepO2 from the starter strain L. helveticus CNRZ32 (Christensson et al. 2002; Chen et al. 2003). Besides cleaving oligopeptides from 7- to 17-residue-long, PepF is also important for protein turnover under conditions of nitrogen starvation in L. lactis (Monnet et al. 1994; Nardi et al. 1997). Other peptidases capable of acting on oligopeptides are the broad-specificity metallopeptidase PepN and cysteine peptidase PepC proteins that were characterized from diverse LAB strains (Table 1). Collectively, these enzymes can remove the N-terminal amino acids from a peptide, the specificity depending on the peptide length and the nature of the N-terminal amino acid residue (Kunji et al. 1996; Christensen et al. 1999). Di/tripeptides generated by endopeptidases, general aminopeptidases, and PepX are next subjected to additional cleavage by the tripeptidase, PepT, and dipeptidases, PepV and PepD (Table 1). These enzymes prefer peptides containing hydrophobic amino acids including leucine, methionine, phenylalanine, or glycine. An enzyme possessing specificity toward di/ tripeptides with N-terminal leucine residues and dipeptides containing proline was biochemically characterized from L. bulgaricus (Klein et al. 1995). Other peptidases with more specific substrate specificities include: PepA, which liberates N-terminal acidic residues from peptides that are three- to nine-residue-long; PepP, which prefers tripeptides carrying proline in the middle position; PepR and PepI, which act on dipeptides containing proline in the penultimate position; PepQ, which cleaves dipeptides carrying proline in the second position; and PepS, which shows preference for peptides containing two to five residues with Arg or aromatic amino acid residues in the Nterminal position (Kunji et al. 1996; Christensen et al. 1999; Fernandez-Espla and Rul 1999) (Table 1).
Stress-related proteolytic systems of LAB The cellular stress responses include the rapid and transient induction of proteolytic activity to cope with the accumulation of abnormally folded proteins caused by a change in the environmental conditions. During industrial
processes the starter bacteria are repeatedly exposed to stress conditions; in a recent report, several stress proteins of starter LAB were identified in the ripening Emmental cheese (Gagnaire et al. 2004). Knowing that several of the stress-inducible proteases are capable of degrading casein in vitro (Katayama-Fujimura et al. 1987; Laskowska et al. 1996), it is tempting to speculate that some of these proteins could play roles in dairy fermentations at the stage where autolysis occurs. While Lon is the primary protease responsible for the degradation of nonnative proteins in Escherichia coli (Gottesman 1996), homologues of Lon were not identified in LAB. Thus, the stress-related proteases characterized to date include FtsH (Nilsson et al. 1994), HtrA (Smeds et al. 1998; Poquet et al. 2000), and the Clp-protease (Frees and Ingmer 1999); it is interesting to note that all of these proteins are required for growth of L. lactis under various stress conditions (Nilsson et al. 1994; Frees and Ingmer 1999; Foucaud-Scheuneman and Poquet 2003). It is noteworthy that clpP of L. lactis, which encodes the proteolytic subunit of Clp-protease, is central in both the proteolysis of misfolded proteins (Frees and Ingmer 1999) and in adjusting the level of key regulatory proteins in the cell (Savijoki et al. 2003). Furthermore, a search for mutations that suppressed the heat sensitive phenotype of a clpP mutant identified the regulatory protein, TrmA, whose inactivation stimulated the proteolysis of nonnative proteins in L. lactis (Frees et al. 2001).
Regulation of the proteolytic systems LAB are likely to respond to changes in nitrogen availability by regulating the activity of the proteolytic system to ensure proper nitrogen balance in the cell. It was suggested that di/tripeptides with hydrophobic residues act as effector molecules in the transcriptional regulation of the Opp system and thereby impact the whole proteolytic system in L. lactis (Kunji et al. 1995, 1996; Detmers et al. 1998). In a more recent study, the expression of six transcriptional units, including prtP, prtM, opp-pepO1, pepD, pepN, pepC, and pepX, were shown to be repressed 5 to 150-fold upon the addition of casein hydrolysate containing 80% peptides and 20% amino acids to the growth medium and the expression was relieved only when cells encountered nitrogen-limiting conditions (Guédon et al. 2001a). Similarly, a proteomics approach revealed that Opp, PepO1, PepN, PepC, PepF, and the putative substratebinding protein (OptS) of a second oligopeptide ABC transporter system are upregulated in L. lactis grown in a medium lacking free amino acids and peptides (Gitton et al. 2005). It is now clear that the transcriptional regulator CodY negatively regulates the expression of several components of the proteolytic system in lactis and that the strength of repression is modulated by the intracellular pool of BCAAs including isoleucine, leucine, and valine (Guedon et al. 2001b; Petranovic et al. 2004) (Fig. 2). In vitro assays indicate that CodY binds to the upstream sequences preceding the opp-operon and that the BCAAs stimulate this binding (den Hengst et al. 2005a). The
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conserved target sequence of CodY, the CodY-box (AATTTTCWGAAAATT), was recently discovered by den Hengst et al. (2005b) by combining a whole genome expression profiling and bioinformatic approaches with classical DNA–protein interaction studies (den Hengst et al. 2005b). This study further demonstrated that CodY regulates its own synthesis and requires isoleucine, leucine, or valine to bind to the CodY box present at its promoter in L. lactis. In summary, in nitrogen-rich medium, the proteolytic system is repressed by CodY and the expression is relieved when cells encounter limiting amounts of BCAAs (Guedon et al. 2001b; den Hengst et al. 2005b) (Fig. 2). The expression of pepF of L. lactis was suggested to be independent of CodY (Gitton et al. 2005); it also appears that the regulation mechanisms of PepO1 and PepC expression are only partly solved because the expression of both of these enzymes is inducible by aeration and respiratory permissive conditions also in the ΔcodY background (Vido et al. 2004). The carbon source in the growth medium was shown to affect the expression of pepP in L. lactis (Guèdon et al. 2001a). This gene is preceded by several potential catabolite-responsive element boxes, suggesting direct control by a CcpA-like regulator. While the heat shock response was proposed to contribute to the regulation of expression of some peptidases in E. coli (Gottesman 1996), in L. lactis, heat stress had no effect on the expression of the peptidolytic system (Guèdon et al. 2001a). Of the stress-inducible proteolytic enzymes, the expression of the proteolytic subunit of Clp protease, clpP, and that of the regulatory subunits clpC, clpE, and clpB are regulated by the stress response regulator CtsR (Varmanen et al. 2000b) that binds to a directly repeated heptad located at upstream regions of target genes (Derre et al. 1999). Furthermore, inactivation of a gene named trmA partially complements a clpP mutation by stimulating Clp-independent degradation of nonnative proteins, indicating that the gene product of trmA is another negative regulator of proteolysis in L. lactis (Frees et al. 2001). Thus, at least three regulators, CodY, CtsR, and TrmA, are involved in regulating the proteolytic activity in L. lactis. The regulatory mechanisms of the proteolytic systems in lactobacilli are much less studied. The peptide concentration in the growth medium was shown to control the PrtH and PrtR biosynthesis in L. helveticus and in L. rhamnosus, respectively (Hebert et al. 2000; Pastar et al. 2003). The PrtH activity of L. helveticus CRL 1062 was shown to be repressed (11 to 32-fold) in a peptide-rich medium in which dipeptide composed of leucine and proline was shown to play an important role in regulation of PrtH activity (Hebert et al. 2000). The addition of peptides into the growth medium also resulted in strong downregulation (17-fold) of the Opp and the DtpT expression in a sourdough starter Lactobacillus sanfranciscensis DSM 20451, whereas the expression of pepT was reduced to a lesser extent (two to fivefold) under the same conditions (Vermeulen et al. 2005). Environmental factors are also known to affect the expression of certain peptidase genes as was recently
demonstrated for a meat starter L. sakei where the pepR expression was repressed during aerobic growth and induced 20-fold upon anaerobic conditions (ChampomierVergès et al. 2002). A recent systematic study on probiotic L. acidophilus using expression profiling with whole genome microarrays revealed that a two-component regulatory system (2CRS) acts as a pleiotropic regulator in controlling the expression of at least 80 genes, including those of the major components of the proteolytic system (Azcarate-Peril et al. 2005). However, the direct regulatory role of 2CRS was not apparent because no simple correlation of changes in gene expression level to proteolytic activity or the loss thereof could be indicated (Azcarate-Peril et al. 2005). To date, only one regulatory protein directly controlling the expression of genes belonging to the proteolytic system was identified (Stucky et al. 1996; Schick et al. 1999). These in vitro studies have demonstrated that PepR1 of L. delbrueckii subsp. lactis binds to the promoter regions of the pepQ, pepX, and brnQ genes, which encode a prolidase, an X-prolyl dipeptidyl aminopeptidase, and a BCAA transporter, respectively. More detailed study on PepQ in L. bulgaricus has indicated that the pepR1 gene encodes a CcpA-like regulator that acts as a transcriptional activator of pepQ expression. The biosynthesis of PepR1 is autoregulated and the regulation depends on the glucose concentration in the culture medium (Morel et al. 1999, 2001). The expression of PepQ was also shown to depend on the carbohydrate composition rather than peptide concentration; this contrasts with the biosynthesis of other components of the proteolytic system in L. bulgaricus (Morel et al. 1999).
Physiological role of proteolytic systems In vitro assays were pivotal in resolving the role of the proteolytic system in amino acid liberation from casein and casein-derived peptides. However, construction of various mutant LAB strains lacking one or several genes has enabled the study of the individual components of the proteolytic system and their coregulation under natural conditions (Kunji et al. 1996, 1998; Christensen et al. 1999; Varmanen et al. 2000a; Garault et al. 2002; Christensen and Steele 2003). Mutant analyses were conducted on L. lactis, L. helveticus, L. rhamnosus, and S. thermophilus. The studies on L. lactis indicate that both the PrtP and Opp are crucial for growth in a medium containing caseins as the sole source of amino acids; whereas the lack of the DtpT has no influence on the growth rate in milk (Kunji et al. 1996; Christensen et al. 1999). Also, in S. thermophilus, the inactivation of Opp results in a growth defect of the bacterium in milk (Garault et al. 2002). In conclusion, Opp is essential for nitrogen nutrition, whereas the di/tripeptide transporters are not directly relevant to the utilization of casein-derived oligopeptides as no significant amounts of free amino acids, dipeptides or tripeptides, are formed by CEPs (Juillard et al. 1995). Instead, the di/tripeptide transporters are suggested to play a role in the regulation
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of the expression of genes involved in nitrogen metabolism (Marugg et al. 1995; Guedon et al. 2001a,b). Numerous LAB strains with mutations in single peptidase genes were also examined for their ability to grow in milk (Kunji et al. 1996; Christensen et al. 1999; Varmanen et al. 2000a). Although some minor changes in growth characteristics were observed, it can be concluded that none of the individual peptidases are essential for growth in milk because their activities can be compensated by the other peptidases (Christensen et al. 1999). In addition to single mutants, multiple peptidase mutants were constructed in L. lactis (Mierau et al. 1996) and in L. helveticus (Christensen and Steele 2003). The L. lactis strain lacking pepX, pepO, pepT, pepC, and pepN grows equally well as the wild-type strain in the complex medium (M17), suggesting that these peptidases have no housekeeping function, for instance, in protein turnover (Mierau et al. 1996). However, the growth deficiency of multiple mutant strains of L. lactis and L. helveticus in milk appears to be more severe when greater number of peptidases are inactivated (Mierau et al. 1996; Christensen and Steele 2003). The availability of whole genome sequences is paving the way for global analyses of gene expression in LAB in vivo. Recent proteomic approaches on LAB exploit the available sequence data for protein identification by mass spectrometry (e.g., peptide mass mapping by MALDI-TOF, i.e., matrix-assisted laser desorption/ionization time-offlight mass spectrometry). Recently, the protein expression profile of L. lactis growing in milk was published by Gitton et al. (2005). Using two-dimensional electrophoresis, Gitton et al. (2005) quantitatively detected about 900 protein spots and identified more than 330 distinct proteins. While 12 of the identified proteins were peptidases (PepA, PepC, PepDB, PepF, PepM, PepN, PepP, PepQ, PepO, PepO2, PepT, and PepV), only four of them (PepC, PepN, PepO, and PepF) were found to be upregulated in milk (Gitton et al. 2005). Of the genes in the proteolytic machinery, the Opp system was found to be strongly upregulated (Gitton et al. 2005). Gagnaire et al. (2004) utilized proteomic tools to characterize the Emmental cheese ripening process. This interesting approach provided information on the peptidases released into the cheese by the starter bacteria L. helveticus and S. thermophilus during ripening. Different peptidases also arose from L. helveticus (PepN, PepO, PepE, and PepQ) and S. thermophilus (PepN, PepX, and PepS). Remarkably, this work suggested the participation of specific peptidases of L. helveticus and S. thermophilus in the degradation of casein-derived peptides during the ripening process of Emmental cheese (Gagnaire et al. 2004).
Technological aspects of proteolysis Proteolysis is considered one of the most important biochemical processes involved in manufacturing many fermented dairy products (Fox 1989b). In cheese manufacture, the proteolysis of casein is thought to play a pivotal
role because amino acids resulting from proteolysis are the major precursors of specific flavor compounds, such as various alcohols, aldehydes, acids, esters, and sulfur compounds (Smit et al. 2005). Bitterness, which results from the accumulation of hydrophobic peptides, i.e., peptides rich with proline, is a serious quality concern facing the manufacture of Gouda and Cheddar cheeses (Smukowski et al. 2003). The specificities of CEPs play an essential role in the production of bitter peptides (Visser et al. 1986; Rodriguez et al. 1996; Broadbent et al. 2002). In addition, the use of LAB strains deficient in peptidase activity have also indicated that peptidases, including PepN, PepX, PepO2, and PepO3, are involved in the degradation of bitter peptides; these peptidases therefore impact the development of the organoleptic quality of the milk product (Meyer and Spahni 1998, Chen and Steele 1998; Chen et al. 2003; Christensen and Steele 2003; Sridhar et al. 2005). It was recently proposed that foodgrade strains of L. lactis expressing L. helveticus CNRZ32 PepO2 and PepO3, in combination with PepN, can be used to reduce bitterness in cheese (Sridhar et al. 2005). For economic reasons, several approaches were exploited to accelerate the ripening process. These have included methods such as elevation of storage temperature, addition of proteinases, the use of bacteriophage-encoded lysin or lytic bacteriophages, and the addition of selected nonstarter LAB or lactobacilli adjuncts to cheese (Fox et al. 1996; Rodríguez et al. 1996; Morgan et al. 1997; MartínezCuesta et al. 1998; Meijer et al. 1998; Madkor et al. 2000). Enriching the L. lactis proteolytic potential by constructing a recombinant starter strain expressing peptidases derived from L. helveticus or L. delbrueckii subsp. lactis under a constitutive or inducible promoter can also be used to accelerate cheese proteolysis and, hence, the ripening process (Wegmann et al. 1999; Guldtfeldt et al. 2001; Luoma et al. 2001; Courtin et al. 2002; Henrich et al. 2002; Joutsjoki et al. 2002; Sridhar et al. 2005). In addition, the cheese trial accomplished by Guldtfeldt et al. (2001) demonstrated that overexpression of, e.g., pepN or pepC in L. lactis subsp. cremoris NM1 background, results in a positive effect on Cheddar cheese flavor and increases the level of specific free amino acids in cheese. However, while it can be concluded that balanced proteolysis is important for flavor formation and especially in prevention of bitterness in cheese, it is the conversion of the free amino acids, rather than proteolysis/peptidolysis, that controls the rate of flavor formation from proteins (Smit et al. 2005). Thus, engineering the proteolytic system alone is hardly the key to accelerating flavor formation in cheese. In addition to the proteolytic systems, the autolysis of LAB starters is considered to be another important element of cheese manufacture because this activity permits the release of cytoplasmic peptidases into the curd, a prerequisite for flavor formation to proceed (Crow et al. 1995). In this regard, a number of studies were sought to control the rate and level of lysis of Lactococcus starter strains; these include phage- and autolysin-based mechanisms and leaky lactococcal starter cultures overexpressing certain L. lactis or L. helveticus peptidases (Buist et al.
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1997; De Ruyter et al. 1997; Guldtfeldt et al. 2001; Tuler et al. 2002; Hickey et al. 2004). The preliminary results suggest the potential of such approaches to accelerate the ripening process and to improve the sensory properties of cheese.
Proteolytic activation of bioactive peptides Besides the importance of the proteolytic/peptidolytic enzymes of LAB to organoleptic properties of the final milk product, certain LAB strains are known to contribute to the liberation of bioactive peptides that are thought to promote health beyond the basic nutrition (Pihlanto and Korhonen 2003; Meisel 2004). In this respect, the casein molecules of milk are of particular interest because they are known to harbor bioactive peptides that are latent until released by proteolysis. To date, LAB ascribed with such activity include strains like L. helveticus CP790, L. rhamnosusGG, L. bulgaricus SS1, and L. lactis subsp. cremoris FT4 (Gobbetti et al. 2002). Several reports have indicated that bioactive peptides, besides existing in naturally ripened cheese and other fermented products, may also be produced in vivo after the intake of milk proteins (Meisel 2004). Manufacturing of such peptides on industrial scale for use as dietary supplements and pharmaceutical preparations is currently receiving increased interest (Korhonen and Pihlanto 2003; Meisel 2004).
Concluding remarks The proteolytic system of the LAB model L. lactis was studied to the extent that a good understanding of the key components involved in casein breakdown, uptake of oligopeptides, and subsequent peptidolytic pathways exists. While the corresponding research on lactobacilli focused mainly on L. helveticus and L. delbrueckii subsp. is also relatively advanced, there are still many unanswered questions, particularly regarding the regulation of proteolytic pathways. With the current knowledge and molecular tools, it has become possible to genetically engineer starter LAB to express desired proteolytic/peptidolytic activity. The increasing number of genome sequences available for different LAB species will allow the integration of functional genomic approaches, including transcriptomics, proteomics, and metabolomics, to define the molecular nature of metabolic networks within LAB and the regulatory mechanisms underlying the physiology traits of these bacteria. This knowledge, aided by the development of novel food-grade genetic tools, will foster the development and screening of new LAB strains, which perform better under different fermentation processes to provide humans with healthy food with appealing texture and flavor.
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